Process for producing 6-amino-penicillanic acid

ABSTRACT

A PROCESS FOR THE PRODUCTION OF HYPOLLAERGENIC 6-AMINOPENICILLANIC ACID BY ENZYMATIC DEGRADATION OF A NATURAL PENICILLIN WITH AN ESCHERICHIA COLI PENICILLIN ACYLASE CHEMICALLY FIXED TO A POLYMERIC SUPPORT.

United States Patent 3,736,230 PROCESS FOR PRODUCING G-AMINO-PENICILLANIC ACID Per Staffan Delin, Villagaten 24; Bertil Ake Ekstrom,

Kummelvagen 26; Lars Solve Nathorst-Westfeld, Kummelvagen 32; BerndtOlof Harald Sjoberg, Kummclvagen 24; and Karl Hugo Thelin,Fornminnesvagen 9, all of Sodertalje, Sweden No Drawing.Continuation-impart of application Ser. No. 799,983, Feb. 17, 1969, nowPatent No. 3,622,462. This application Nov. 22, 1971, Ser. No. 200,826

Int. Cl. C12d 1/02 US. Cl. 19536 P Claims ABSTRACT OF THE DISCLOSURE Aprocess for the production of hypoallergenic 6-aminopenicillanic acid byenzymatic degradation of a natural penicillin with an Escherichia colipenicillin acylase chemically fixed to a polymeric support.

This application is a divisional and continuation-in-part application ofour application Ser. No. 799,983, filed Feb. 17, 1969, now US. Pat. No.3,622,462.

This invention relates to an improved method for the production of6-aminopenicillanic acid (6-APA) by enzymatic degradation of apenicillin and to improved method for the production of penicillinsespecially for that of hypoallergenic penicillins. It further relates toan improved method for the production, purification and isolation of anenzyme preparation capable of removing the side chains of certainpenicillins under formation of 6APA. In particular it refers to theproduction of an enzyme preparation from Escherichia coli and its usefor the production of 6-APA from benzylpenicillin.

It is known that many microorganisms, both bacteria and fungi, produceenzymes, which can hydrolyse the amide bond in the 6-position ofpenicillins and which are generally termed penicillin acylases oramidases (for a review, see J. M. T. Hamilton-Miller BacteriologicalReviews (1966) 761). In the present industrial production of 6-APA,suspensions of E. coli cells containing an acylase or amidase arepreferably used. These processes, however, have several drawbacks. Sincethe enzyme is largely intracellular, the penicillin must first, ofnecessity, penetrate the cell bodies in order to react with the enzymewhich results in a slower reaction. The strain used may also containother enzymes which inactivate the penicillin, or the 6-APA formed, bysplitting the fi-lactam bond or which may contaminate the cell culturewith the organisms that produce such enzymes. As the acylase constitutesonly a very small portion of the cell content a process using wholeorganisms also has a practical disadvantage in that it involves largeamounts of material that are inactive in the process.

A further disadvantage experienced by the use of whole organisms is thatan extra step must be added to the series of processes leading to theproduction of semisynthetic penicillins, namely, the separation (e.g.,by filtration) of the organisms from the reaction fluid in which theoriginal side chain has been split off from biosynthetic penicillins. Afurther disadvantage is the loss of penicillanic acid caused partly byadsorption to the cells, and partly by degradations induced by materialsproduced during the cell metabolism. Still another factor tending togive rise to problems associated with the use of whole cells for thepreparation of 6-aminopenicillanic acid from biosynthetic penicillins isthat separation of the acid from other products of the split offreaction is made quite difficult. Thus Batchelor et al. (Lancet I (1968)1175) have reported that 6-APA obtained by the use of whole cells maycontain proteinaceous impurities capable of eliciting dangerousallergenic reactions in man and animal. When penicillins are made fromsuch contaminated 6-A'PA the impurities may be retained in the productsand be responsible for many of the allergic reactions observed withthese penicillins (G. T. Stewart, Amer. Heart. J. (1968) 429). In orderto remove these impurities, the 6-aminopenicillanic acid or thepenicillins prepared therefrom must be subjected to additionalseparation processes including, e.g., dialysis or gel filtration(Canadian Pat. No. 771,- 662). Hereby large proportions of the 6APA orof the penicillin are lost as demonstrated by a recovery of only 12% ofbenzylpenicillin (British Pat. 1,078,847) and of 56% ofphenoxymethylpenicillin (British Pat. 1,114,311).

All these obstacles can be avoided when a cell free or purified enzymepreparation according to the present invention is used. Thus the 6-APAformed by using a cell free or purified enzyme preparation according tothe present invention for the splitting of the amide bond in the6-position of an enzymically deacylable natural or biosyntheticpenicillins are obtained in good yields and are essentially free fromproteinaceous impurities. The term natural or biosynthetic penicillinsas used in this specification refers to such penicillins where the sidechain is derived from a monosubstituted acetic acid. Such penicillinsmay be obtained by fermentation in substrates containing the appropriateacetic acid or a suitable derivative thereof. When the 6-APA prepared inthis way is acylated hypo-allergenic penicillins are obtained in goodyields without any additional purification. In the literature severalattempts to prepare purified enzyme preparations from E. coli strainshave been recorded. Borkar et al. (Hindustan Antibiotics Bull. 4 (1961)48, 152) treated cells suspended in a phosphate buflfer with ultarsonicwaves and achieved a twenty-five fold purification by fractionatedprecipitation and column chromatography. Holt and Stewart (Nature 201(1964) 824) obtained an enzyme preparation of low purity byfreeze-drying and dialyzing filtered culture broth. Szentirmai (ActaMicrob. Acad. Scient. Hung. 12 (1966) 395) obtained a fortyfoldpurification of an E. coli enzyme by ammonium sulphate precipitation,followed by calcium phosphate gel adsorption and DEAE cellulosechromatography of a phosphate bufier extract of E. coli cells treatedwith ultrasonic waves.

Sakaguchi and Murao (Japanese Pat. 26,050/64) obtained a moderate yieldof a purified enzyme preparation from E. coli by extracting the cellswith borate buffer for an extended period of time or for a shorter timein combination with ultrasonic waves treatment. From the extract theenzyme could be obtained in solid form after precipitation with ammoniumsulphate, dialysis and freezed-drying. Johnson and Hardcastle (US. Pat.3,297,- 546) obtained a solution of an enzyme from E. coli by treatingthe cell culture with a compound MX usually Ca(NO and a quaternaryammonium compound, filtering off the cells and suspending them in waterfor some hours, and then removing the cells by filtration withsubsequent treatment of the filtrate with activated carbon.

It is further known that enzymes contained in bacterial cells can bereleased on high pressure extrusion of cell suspensions through a smallorifice. Duerre and Ribi (Appl. Microbiol. 11 (1964) 467) studying othertypes of enzymes than penicillin amidases found in E. coli observed thathigh pressure, in excess of 15,000 p.s.i. had to be used to obtainmaximal release of the enzymes. At such pressures, however, the cellwalls were also fragmented and large portions of the cell material wassolubilized. Frazer (Nature 167 (1951) 33) found that E. W cells couldbe ruptured on a small scale when they were expelled from a bomb by agas pressure of 500-900 p.s.i.

We have now found that the intracellular penicillin acylase in E. colican be extracted from the organism into Water in large production scaleafter the cells, or suspensions of them in water or a mixture of Waterand organic solvents, have been rapidly released from an appliedpressure of at least 500 p.s.i. but not exceeding the pressure whereexcessive disruption of the cell occurs.

Thus, the present invention in one aspect provides a process forproduction of penicillin acylase preparation from E. coli comprisingfermentation in a known way of an E. coli strain producing such anenzyme, removing the culture fluid and rapidly expelling the cellmaterial through a narrow orifice or slit by application of pressure ofat least 500 p.s.i. but not exceeding the pressure where excessivedisruption of the cell occurs. Still at 3,000 p.s.i. the cells are notexcessively disrupted. The expelled material is then stirred with watereventually with addition of an organic solvent and/or a base, likesodium hydroxide or triethylamine, to dissolve the enzyme.

In one embodiment of the present invention the removal of the culturefluid and the expulsion of the cell material are effected simultaneouslyin a self-cleaning centrif-ugalseparator, operated at a temperaturebetween -50" C., preferably at -40 C. where the separated cell materialintermittently is expelled within 0.05-1.0 sec., preferably within0.1-0.5 sec., through a periferal slit with a width of 0.1-1.5 mm.,preferably 0.3-0.7 mm. by application of pressure of 500-2,000 p.s.i.,preferably 900-l,100 p.s.i. If desirable, the broth is saturated with anorganic solvent having low solubility in water such as butylacetate,isobutylacetate or amyl acetate in order to kill the organism, and toaid the process in the separator. Likewise it is possible, if desirable,to wash the cell material with water in the separator. The expelled cellmaterial is stirred at 10-50" C., preferably at -40 C., for 0.10-5.0hrs., suitably 0.25-3.0 hrs. and preferably 0.25-1.0 hr. with anefficient stirrer to dissolve the enzyme, and possibly with addition ina concentration of 1.0-5.0% of a water immiscible organic solvent suchas methylisobutyl ketone, butylacetate, isobutylacetate, amyl acetate,benzene, toluene or chloroform. In order to facilitate the extraction ofthe enzyme from the expelled cell material an inorganic base, such assodium hydroxide, potassium hydroxide or ammonia or a tertiary organicbase such as triethylamine or N-ethylpiperidine, may be added to themixture to adjust and sustain the pH at a value between 6.5 and 9.0,preferably between 7.0 and 8.5.

The enzyme solution thus obtained can, possibly after dilution withwater, be freed from any remaining cell material and other solidimpurities by common processes such as filtration or centrifugation, andeventually by a combination of both processes and possibly with theaddition of decolorizing, clarifying and filtering aid agents such asactivated carbon, aluminia, cellulose powder, diatomaceous earth orother solid, weak adsorbing agents.

"One preferred methodis to remove most of the cell material bycentrifugation and to filter the supernatant.

Additional purification of the enzyme can be obtained by acidificationof the aqueous solution to pH 3.0-6.0,

preferably to 4.0-5.0, and then adjusting the solution to pH 7.0-8.5with removal of the precipitatedinactive material by filtration aftereach adjustment of pH and finally readjusting the pH to its originalvalue.

The penicillin acylase contained in the cell free, and if desired,partly purified aqueous solutions referred to above can be precipitatedby treatment of the solutions with agents, like tannins, which formsparingly soluble complexes with proteins. In one preferred form of thisprocess the enzyme solution is treated at pH 4-6, suitably at pH 4.5-5.5with tannin to a final concentration of 300- 900 p.p.m. in presence ofchelating agents, like ethylenediamine tetraacetic acid, formingcomplexes with iron ions. The enzyme-tannin complex formed my beisolated in a common way, eg by filtration or centrifugation. It may bewashed and dried and freed of water, e.g. by drying, especially byfreeze-drying, or by treatment with 4 water-miscible organic solventslike acetone. The tannin complex is a suitable form for storage andtransport of the enzyme. It can also be used directly for the removal ofthe side chain of penicillins like benzylpenicillin.

The penicillin acylase contained in the tannin precipitate may bereleased into aqueous solution by dissolution of the complex in water atpH 7-9, preferably 7.5-8.5. Alternatively the complex may be treatedwith a mixture of water and a water immiscible solvent like n-butanol atpH 4-7, preferably 4.5-5.5. A third method of removing the tanninconsists in treating the enzyme-tannin complex, suspended in Water withan anionic ion exchanger, like DEAE-cellulose, which binds the tanninand releases the enzyme into water. The amounts of water necessary forthese operations are far less than those present in the original enzymesolutions and in this way a considerable concentration of the enzymaticactivity is achieved.

In a further aspect of the invention the penicillin acylase contained inany solution referred to above may be concentrated and purified with theaid of an ionv exchanger. In a preferred form the enzyme is adsorbedorganic ions and low molecular weight impurities can be removed from theenzyme solutions by dialyzation against water. Alternatively thesolutions if necessary after concentration by evaporation at atemperature below 50 C. to a suitable concentration of 25-100 mg. dryweight per ml., are submitted to gel filtration.

The aqueous enzyme solutions thus obtained at any of the previous stagesare highly suitable to be used directly for the removal of the sidechains of natural penicillins, especially for removal of the side chainof benzylpenicillin.

In a further aspect of the present invention the enzyme as heretoforeprepared is isolated from aqueous solution in the solid state byfreeze-drying, float-drying, spray-drying or concentration in vacuo orby precipitation according to known procedures. The isolation processresults in a solid enzyme possessing a high specific activity and whichis a very suitable form for storage or transport. The

use of such solid, water soluble enzyme preparations also has severaltechnical advantages as it makes possible work with more concentratedsolutions than those utilized by the known processes which employ cellsuspensions.

processes, using suspensions of E. coli cells.

We have also found that the 6-arninopenicillanic acid prepared accordingto this invention contains very little if any of the proteinaceousimpurities with allergenic properties obtained when suspensions of E.coli cells are used for its production. This is of great technicalimportance as it is thus possible to prepare penicillins withhypoallergenic properties directly from the 6-APA prepared according tothis invention without any additional purification processes. Examplesof penicillins with hypo-allergenic properties which may be prepared inthis way are: a-phenoxyethylpenicillin, a-phenoxypropylpenicillin,2,6-dimethoxyphenylpenicillin, 3 (o chlorophenyl)-5-methyl-4-isoxazolylpenicillin, a-carboxy-benzylpenicillin,a-azidobenzylpenicillin and ot-aminobenzylpenicillin.

The penicillin acylase of E. coli can also be used for removing the sidechain of penicillin esters especially of esters of benzylpenicillin asdescribed in British patent application No. 33,734/67. We have now foundthat the enzyme preparations obtained according to this invention arevery suitable for this process and are more efficient than suspensionsof E. coli cells previously used. Furthermore we have also found thatthe enzyme preparations according to this invention are superior to thepreviously used cell suspensions in the enzymatic synthesis ofpenicillins from 6-aminopenicillanic acid and side chain precursor (W.K. Kaufmann et al., Antimicrobial Agents, Ann. 1960, 1).

The purified enzyme preparations obtained according to this inventionare suitable starting materials for chemical modification of the enzymeresulting in products with better properties with respect to activityand/or technical performance. Thus the enzyme can react with activatedpolymeric materials, such as, e.g. polysacharides treated with cyanogenbromide, to give products where the enzyme is fixed to a polymericsupport. The enzymatic activity is retained in such products which areof advantage to use for the splitting of penicillins as they can berecovered from the reaction solution by simple means, e.g. byfiltration. The polymeric enzyme-preparation can also be used in columnsfor continuous production of 6-APA by passing solution of penicillinsthrough the column.

A preferred method for chemical modification of the acylase is bycovalent coupling to insoluble activated polysaccharides, for instance,dextrane, Sephadex, a-glucan or Sepharose. The method comprisesequilibrating the polysaccharide with water at room temperature, coolingof the mixture and, thereafter, adding of sodium hydroxide followed by acyanogen halide, for instance cyanogen chloride or cyanogen bromide. Thecyanogen halide is added, with stirring, suitably at a temperature offrom C. to about 25 C., preferably at a temperature of from 0 C. toabout 10 C., whereupon the activated polymer is separated and washedwith water. Coupling of penicillin acylase to the active wet polymer canbe effected by reacting the components in a water suspension, suitablyat a temperature of from about 2 C. to about 10 C., preferably at atemperature of from about 3 C. to about 5 C., suitably during a timeperiod from about 2 to about 30 hours, preferably during from about toabout 26 hours, at pH 7-9.5, preferably at pH 8.8- 9.0. This pH-valuecan be effected by adding a suitable base e.g. sodium hydroxide or abuffering substance, for instance borax. Thereafter the polymer-acylaseis separated.

A preferred method for production of 6-aminopenicillanic acid by use ofacylase covalently coupled to a polymer, for instance a polysaccharide,comprises adding to a suspension of the acylase-polymer in water,containing a suitable buffering substance, a biosynthetic penicillin,preferably benzylpenicillin, in a concentration of from about 1 to about10%, preferably from about 2 to about 5%, and subsequently incubatingthe mixture, with stirring, at a temperature of from about 30 C. toabout 45 0., preferably at a temperature from about 35 C. to about 40 C.until the enzymatic reaction has ceased. During the reaction the pH isto be kept at 6.5-8.2, preferably at 7.3-7.8, by continuous addition ofa solution of an alkaline compound, for instance sodium hydroxide orpotassium hydroxide. The acylase-polymer is separated by filtration orcentrifugation, washed with water and, thereafter, without demonstrableloss in enzymatic activity, ready for use in another splitting reaction.From the water solution obtained the acid which is split off in theenzymatic reaction, for instance phenylacetic acid frombenzyl-penicillin, can be extracted by means of a suitableWater-immiscible organic solvent for instance methylisobutyl ketone,ethyl ether, butyl acetate or isoamylacetate at pH 1.5-4, preferably2-3. The organic layer is separated and the water phase neutralized topH 7-8, preferably 7.4-7.7, by means of alkali, for instance sodiumhydroxide or potassium hydroxide, whereupon the solution is concentratedin vacuo, preferably to a concentration of at least 10% of6-aminopenicillanic acid. The concentrated solution is acidified to pH4-4.6, preferably to 4.3-4.4, by means of a mineralic acid, for instancesulphuric acid, hydrochloric acid or nitric acid. The precipitatedcrystalline 6-aminopenicillanic acid is isolated and dried forsubsequent use as a starting material in production of semisyntheticpenicillins according to known methods.

It is also possible to make semisynthetic penicillins directly from theoriginal solution obtained in the enzymatic splitting of penicillinwithout previously extracting the acid originating from the side chainand isolating of the 6-aminopenicillanic acid. The G-aminopenicillanicacid and/or the semisynthetic penicillin compounds isolated followingenzymatic splitting of penicillin by use of polymer-bound acylase showvery low contents of proteinaceous and other high molecular impuritieswith allergic properties compared to the compounds obtained by means ofsuspensions of acylase-producing E. coli cells.

The preparation and purification of a cell free enzyme product, thepreparation of 6-aminopenicillanic acid from natural penicillins (e.g.benzyl penicillin) and the preparation of synthetic penicillins from6-aminopenicillanic acid is illustrated in the following examples.

EXAMPLE 1-PRODUCTION OF E. COLI BACTERIAL CELLLS Corn steep liquor (3kg), soybean oil ml.), paraffin (12 ml.) and cetanol (3 ml.) in water(1501.) was adjusted to pH 6.0 with 45% sodium hydroxide ml.) and thensterilized at 124 C. for 30 minutes in a fermentation seed tank. Thesolution was inoculated with 100 ml. of a 20-24 hr. culture of E. coli(Astra 1339) and incubated at 25 C. with aeration and agitation for 18hrs.

Corn steep liquor (300 kg.), soybean oil (13.8 kg), parafiin (1.22 kg.),cetanol (0.3 kg.), phenylacetic acid (21 kg.) and sodium chloride (112kg.) in water (14,000 1.) was adjusted to pH 6.6 with 45 sodiumhydroxide (40 kg.) and the solution sterilized in a fermentation tank at124 C. for 30 minutes. The solution was cooled and inoculated with theabove mentioned seed culture and subsequently incubated at 25 C. withagitation and aeration. Twenty-four hours after the inoculation and whenthe pH had risen to 8.2, the bacterial cells were killed by the additionof butyl acetate l.) and the mixture was subsequently cooled.

EXAMPLE 2-ISOLATION AND PURIFICATION OF ACYLASE (a) For isolation of thebacterial mass the mixture was separated in a self-cleaning centrifugalseparator (De Laval, type BRPX 213-35S). The bacterial cell paste wascollected in 100-120 kg. portions. To each portion was added 3.0% ofmethylisobutyl ketone and the mixture was then homogenized by means ofan Ultra Turrax stirrer (type T 110/2 M) for 25 minutes. The total yieldof bacterial mass was 325 kg.

Analysis of the enzyme in the different production steps gave thefollowing figures in acylase units determinative of the amount of enzymeremaining during the particular step (one acylase unit corresponds tothe amount of en zyme capable of splitting in 1.5 hours at pH 8.5 and 37C. an amount of benzylpenicillin equivalent to 1 mg. of6-aminopenicillanic acid):

Production fermentation culture u./ml 5 Supernatent liquid u./ml 0.33Centrifugal separator liquid efiluent u./ml 0.28 Bacterial cell pasteu./g 212 Supernatent liquid in paste, before homogenization u./g 75Supernatent liquid in paste, after homogenization u./g 123 (b) Bacterialcell paste obtained as in Example 2a was used in experiments on enzymesolubilization. To 100 g. of cell paste 100 ml. of deionized water wasadded.

The suspension obtained had a pH of 5.9 and was submitted to a series oftreatments as follows:

(I) The suspension was centrifuged at 5,000 G (centifugal force measuredin multiples of gravity) for 20 min. and the enzyme activity in thesupernatent fluid was determined.

(II) The suspension was submitted to homogenization by means of an UltraTurrax stirrer for 5 min. During this treatment the sample was cooled inan ice bath in order to avoid temperatures higher than 35 C. in thesample. The sample was then centrifuged as in I and the activity in thesupernatent was determined.

(III) The pH of the suspension was adjusted to 8.5 with NaOH withstirring whereafter the sample was homogenized and centrifuged as aboveand the activity determined in the supernatent phase.

(IV) 3% methylisobutyl ketone was added to the suspension, the pH ofwhich was adjusted as in experiment III. This sample was then submittedto homogenization and centrifugation as above and the enzyme activitydetermined in the supernatent phase.

The solubilization process may be demonstrated by the following table.

ENZYME SOLUBILIZATION (c) Homogenized bacterial paste (175 kg, activity190 u./ g.) was diluted with water (175 kg.) and Hyflo (22.7 kg), Celite505 (22.7 kg.), Fibraflo (10.2 kg.) added with agitation of the mixture.The water phase was separated by filtering on a Funda filter and thefilter cake Washed with water (50 1.). The combined solutions (290 kg.)had an activity of 48 u./ g.

(d) Homogenized bacterial paste (1 kg., activity 212 u./g.) was dilutedwith Water (2.01.) adjusted to pH 8.5 'by means of 0.5 N sodiumhydroxide (220 ml.) and then stirerd at 20 C. for 3-0 minutes. Themixture was centrifuged at 13,200 G (Serwall, Type RC-Z, AutomaticSuperspeed Refrigerated Centrifuge) for 30 minutes at C. The supernatentliquid was decanted, clarified by filtration giving 2.7 kg. solutionwith an activity of 50 u./g. Resuspension of the sediment in water (1l.) twice for 5 minutes at pH 8.5 followed by centrifugation gave anadditional enzyme solution (2 kg, activity 11 u./g.). This correspondsto a total yield of activity of 74% of Water soluble acylase.

(e) Clear acylase solution (2 kg, activity 50 u./g.) 7

running tap water. The dialyzed solution was lyophilized yielding 20.5g. of solid acylase, activity 2,400 u./ g.

(h) Clear acylase solution (245 kg, activity 48 u/ g.) obtainedaccording to Example 20 was stirred and adjusted to pH 4.5 by means of9-M acetic acid (2.2 kg). The agitation was continued for 1 hr. andthereafter the mixture was filtered and the clear solution adjusted topH 8.0 with 7-M ammonia (4.5 kg.) during agitation. After 1 hr. themixture was filtered and the solution adjusted to pH 5 by means of 9-Macetic acid (2.0 kg). The activity of the solution (220 kg.) was 47.5u./g.

(i)' Supernatent liquid (500 g., activity 52 u./g.) obtained as inExample 2d was adjusted to pH 4 and stirred for 15 minutes at 0 C. inorder to precipitate protein impurities. After centrifugation andadjustment of pH to 7.5 the clear solution obtained (500 g., activity 40u./g.) was dialyzed against tap water overnight and freeze-dried to give4 g. with an activity of 4,900 u./ g.

(j) Homogenized bacterial paste (1 kg. activity 128 u./ g.) wascentrifuged at 113,200 G (Serwall, Type RC-Z, Automatic SuperspeedRefrigerated Centrifuge) for 30 minutes at 0 C. The supernatent liquid(500 g., activity =u./g.) was decanted, clarified by filtration and usedas such for enzymatic splitting of penicillin to produce6-aminopcnicillanic acid.

EXA MPLE 3PREPARATION OF ACYLASE WIIH TANNIN Acylase solution pHfractionatedaccording to Example 2h (185 kg., activity 47.5 u./g.) wasagitated and treated with ethylenediamine tetraacetic acid (50 g.)followed by a solution of tannin (166 g.) and sodium sulphite (55 g.) inwater (5.5 1.). After stirring for 1 hr. Celite 505 (1.5 kg.) and sodiumsulphite (700 g.) was added and the suspension filtered yielding 3,950g. of wet solid tanning-acylase precipitate with an activity of 1,930u./ g.

EXAMPLE 4-RECOVERY OF AOYLASE FROM THE TANNIN PRECTPITATE (a) Wettanning-acylase precipitate acording to Example 3 (30.4 g., activity1,930 u./ g.) Was treated with cold acetone (30 ml., l10 to 30 C.) whichwas added slowly with stirring. After the precipitate had partlydissolved an additional portion of cold acetone (200 ml.) was added atonce and the mixture filtered. The solid was washed with cold acetoneand dried in air at room temperature overnight. Yield 15.4 g., activity3,650 u./ g.

(b) Wet tannin-acylase precipitate (3,750 g., activity 1,930 u./g.)according to Example 3 was suspended into 0.1 molar ammonium acetate, pH5.0 (5.0 l.) and butanol (2.5 1.). Sodium sulphite (50 g.) was added andpH adjusted to 5.0 by means of acetic acid. The stirring Was continuedfor 20 minutes and the mixture filtered. The water phase (6,650 ml.,activity 800 u./ml.) was separated.

(c) Acylase solution (1,000 ml., activity 49' u./ml.), pH-fractionatedas in Example 2h, was treated with tannin according to Example 3. Thetannin-acylase precipitate was filtered and suspended with stirring into0.1 M ammonium acetate buffer, pH 8.0 ml.). The stirring was continuedfor 1 hr. and pH controlled and adjusted to 8.0. The mixture wasfiltered giving 100 ml. of an acylase solution containing 220' u./ ml.

(d) Wet tannin-acylase precipitate suspended in buffer solution, pH '8,(1 00 ml.) as in Example 4c was treated with DEAE-cellulose (2 g.,capacity 1.0 m.-eq./g.). The mixture was stirred for 15 minutes andfiltered giving an acylase solution (90 ml.) containing 494 u./ml.

(e) {Wet tannin acylase precipitate (36.6 g.) activity 1,250 u./g.)obtained as in Example 3 was suspended into 0.2 M ammonium acetatebuffer, pH 8 (150 ml.) and adjusted to pH 8.0. After stirring for 10minutes butanol (60 ml.) was added followed by 9-M acetic acid to pH5.0. The stirring was continued for 10 minutes and the mixture filtered.The water layer (1 66 ml., activity u./ml.) was separated.

9 EXAMPLE LYOPHILIZATION OF TANNIN- ACYLASE PRECIPITATE Wettannin-acylase precipitate (86.1 g., activity 1,600 u./g.) obtained asin Example 3 was cooled to 30 C. and dried in vacuo at 25 C. and 0.1-0.2torr. The yield was 28.5 g. of a dry product with an activity of 2,340u./g.

EXAMPLE 6ISOLATION OF THE ENZYME BY DRYING (a) Spray-drying.Clearacylase solution obtained as in Example 2h (5 1., activity 47.5 u./ml.)was spray dried at an air temperature of l1l5 C. at the inlet and 7075C. at the outlet during a period of 1 hr. Yield 85 g. activity 380 u./g.

(b) Float-drying.Cellulose powder (100 g.) was mixed with acylasesolution according to Example 412 (200 g., activity 162 u./g.). Theresulting mixture was float dried in a stream of air at 40 C. for 90min. 93.2 g. of a dry powder was recovered with an activity of 284 u./g.

EXAMPLE 7PURIFICATION OF THE ACYLASE ACTIVITY BY ION EXCHANGE CHROMATOG-RAPHY (a) Acylase solution (5,000 ml., activity 48 u./ml.) obtained asin Example was adjusted to pH 4.6 by means of 9-M acetic acid. Afterstanding overnight in cold the mixture was filtered and the clearsolution submitted to an ion exchange column (Sulphoethyl Sephaldex C50, diameter 8 cm., length cm. in 0.1 M ammonium acetate, pH 4.6). Thecolumn was washed with the ammonium acetate buffer, pH 4.6 (650 ml.).The acylase was eluted with 0.2 M ammonium acetate bufiFer pH 8.0. Theactivity was obtained in 640 ml. with an activity of 326 u./ml.

(b) A purified solution of acylase (5,550 ml., activity 800 u./ml.)obtained according to Example 4b was adjusted to pH 4.6 and treated inthe same way as in Example 721. 2,350 ml. was obtained with an activityof 1,610 u./ml.

(c) Acylase solution, purified on SE-Sephadex as described in Example 7b(250 ml.) actiivty 1,610 u./ml.) was cooled to 40 C. and dried in vacuoat 0.10.2 torr by a temperature of C. The yield was 2.11 g. of purifiedacylase with an activity of 190,000 11./ g.

'(d) Acylase solution, purified as in Example 7b (230 ml., activity 1610u./ml.) was concentrated by evaporation in vacuo to a volume of 24 ml.15 ml. of the concentrate, activity 15,000 u./ml., was submitted to aSephadex G 75 column (diameter 2.5 cm., length 100 cm.). The acylase waseluted with deionized water. The acylase was obtained in 108 ml.,activity 1,710 u./ml. Freeze-drying of this solution gave 1.8 g. with anactivity of 92,500 u./g.

(e) Acylase preparation (72 mg.) obtained from a Sulphoethyl SephadexG250 column according to Example 7a and freeze-dried was dissolved in0.005 M Na-phosphate bufier pH 6.30 (2.7 ml.) and dialyzed against thesame butter and then applied to a column containing carboxymethylcellulose (Whatman CM-32, small ion capacity 1 meq. per g.; columnlength 13.5 cm., diameter 0.9 cm.) previously equilibrated with thebuffer. The column was eluted with the following buifers: 0.005 MNa-phosphate buffer pH 6.30 (about 17 ml.), 0.02 M Naphosphate buffer pH6.35 (about 17 ml.), and 0.1 Naphosphate butter pH 6.35.

Fractions of 1.3 ml. were collected and the extinction at 280 mmeasured. The total enzyme activity was eluted with the 0.1 M butter andcontained only 10% of the original protein content. About 90% of thetotal extinction were found as inactive protein in fractions containing0.005 M bufier.

(f) A solution obtained according to Example 2b (100 ml.) was dialyzedagainst distilled water and then against 0.005 M phosphate butler pH 6.0for 24.5 hours.

0 Diethylaminoethyl. cellulose (Whatman DE-32, small ion capacity 1.0meq. per g.) (12.5 mg./ml.) equilibrated with the above mentioned butterwas added. The ion exchange cellulose was separated after minutes. Nodecrease of acylase activity was observed but the extinction at 280Ill/.1. was only 10% of the previous value. The enzyme activity was thenadsorbed by carboxymethyl cellulose (Whatman CM-32) treated with HCl andwashed or equilibrated with 0.005 M sodium acetate pH 5.5. In both casestwo different amounts of ion exchanger were used namely 12.5 mg. and 3.2mg. per ml. respectively. After separation of the ion exchanger theenzyme activity was eluted after dispersing the ion exchanger in waterby increasing the pH to 6-7 and the ionic strength by addition of 0.1 Mphosphate butter. The yield was about (g) A solution obtained accordingto Example 2h ml.) was dialyzed against distilled water and then against0.005 M phosphate butler pH 6.0 for 22 hours. The solution was treatedtwo times with diethylaminoethyl cellulose (Whatman DE32) (12.4 mg. perml.) equilibrated with the above mentioned buffer. The solution was thentreated with the same ion exchanger (13 mg. per ml.) in OH-form withoutloss of acylase activity. The ion exchanger was separated 30 minutesafter the addition of the same. After the last addition the pH hadincreased to 8.31. The enzyme was then absorbed completely afteraddition of the ion exchanger in OH-form (50 mg. per ml.). The pH was9.11. After separation of the ion exchanger and dispersion of it inWater the enzyme was eluted by adjusting the pH to about 6. The yieldwas above 60% and the purity of the enzyme measured as the ratio ofactivity to extinction at 280 ml was increased twentyfold.

(h) A solution obtained according to Example 2c (100 ml.) was partiallydesalted by dialysis against distilled water for one hour and thentreated with carboxymethyl cellulose in the H+-form (Whatman CH-32 smallion capacity 1 meq. per g.) (about 40 mg. per ml.). The pH wascontrolled to be 4.0-4.5. The ion exchanger was separated and theabsorbed acylase eluted by increasing the pH to about 6 and the ionicstrength by addition of phosphate bulfer.

(i) The procedure according to Example 7h was repeated but as startingmaterial a solution obtained according to Example 2h was used.

(j) A solution (100 ml.) obtained according to Example 2c was partiallydesalted and deproteinized by treatment with about 25 mg. per ml. ofcarboxymethyl cellulose (Whatman CM-32) in the H+-form and then with thesame amount of diethyl aminoethyl cellulose (Whatman DE32) in theOH-f0rm. The pH was controlled to fall within the interval 57. (Thetreatment may be repeated but should be stopped before substantialamounts of acylase are absorbed.) The enzymes was then adsorbed bycarboxymethylcellulose and eluted as in Example 7h.

(k) A solution obtained according to Example 2c (100 ml.) or 2h wastreated as in Example 7j except that no diethylaminoethyl cellulose wasused.

EXAMPLE 8CONCENTRATION-PUR\IFICATION OF THE ACYLASE ACTIVITY BYULTRAFILTRA- TION A solution obtained according to Example 2c (10 ml.)and partially desalted and deproteinized by treatment with ion exchangeras described in Example 7j, but omitting the last step comprisingadsorbance by carboxymethylcellulose, was diluted with distilled water(40 ml.) and pressure filtrated through a membrane completely retainingmolecules having a molecular weight of more than 50,000 (Diaflo membranefrom.- Amicon Corporation, Cambridge, Mass., U.S.A.). The concentrate (2ml.) containing the main part of the acylase was freeze-dried.

EXAMPLE 9PR-ECIPITATION OF THE ACYLASE ACTIVITY BY MEANS OF AMMONIUMSULPHATE (a) To a purified solution of acylase obtained as in Example 4b(1,000 1111., activity 358 u./ml.) was added 12 (d) Experiment b wasrepeated with the Sephadex G 25 replaced with cellulose powder(Macherey, Nagel & Co., MN-300; 0.1 g.). The yield Was 0.35 g. ofpolymer with an specific activity of 261 u./ g.

ammonium sulphate (570 g.) The mixture was stirred un- 5 EXAMPLE13PRODUCTION OF 6-AMINO- til the salt had dissolved. After standing at 5C. for 2 PENICIILANIC ACID hrs. the precipitate formed was filtered. Theyield was 52.5 To an enzyme solution (283 g activity 42 was Of Wetcompound Wlth an actwfty of added a solution of benzylpenicillin (20 g.)as potassium (b) The wet compound obtained as n Example 9a Salt in Water(317 3 activity 5160 W dried m air at 25 10 The mixture was incubated at37 C. and the pH kept overnight- Yleld 19-85 acm'lty 7,580 at 7.8 by theperiodic addition of 2.5 N sodium hydroxide. EXAMPLE 10 PURI FICATION OFTHE ACYLASE After 6 hours the reaction mixture contained 10.7 g. ofACTIVITY BY DIALYSIS 6-aminopenicillanic acid (92%) and was filtered.The pH of the solution was adjusted to 3 with 5 N hydrochloric yi501M101! 1111-, i y 48 9 obtal'ned acid and extracted with a half volumeof methylisobutylaccording Example was dlalyzfijd agalnst Pf ketone toremove the residual benzylpenicillin and the Ovemlghtf dlalyzed sQlutlonlyophlllzed phenylacetic acid side chain, which is split off during theas 111 Example Yleld with an actlvlty of 8:000 formation of the6-aminopenicillanic acid. After the exu./ g. 20 traction the watersolution was separated from the methyl- EXAMPLE 11PUR[FICATION OF THEACYLASE lsfitylkewne- ACTIVITY BY GEL FILTRATION e pH of the watersolution was adJusted to 7.5 and the volume reduced to 120 ml. in vacuo.The concentrated Acylase SOhlfiOH, Obtainfid according to p 41) Solutionwas cooled to 5 C. and filtered. When the filtered 'a activity 166 Wasconcentrated y p S lution was acidified to pH 4.3 with 5 N hydrochloriction in VaCllO t0 VOlllme 0f 9 0 Submitted to gel filtraacid thecrystalline 6-aminopenicillanic acid precipitated. 011 P G column 3 X 80and eluted After filtration the crystals were washed with a little hdeionized Water- The activity, Was cold water followed by dry acetoneand dried in vacuo CUVeI'ed in 80 I111- Containing 0f the pp mateto givea yield of 8.84 g. of 6-aminopenicillanic acid. The rial, measured on anadsorption at 280 m basis. purity of the product was 95% and the totalyield calcu- EXAMPLE 12 COUPL1NG OF E. C 0L1 ACYLASE lated on thebenzylpenicillm Was 72%.

To POLYMERS EXAMPLE l4-PREPARATION OF 6-AMINOPENI- (a) An agarosesolution (Sepharose 4B, 4%, 2. 5 ml.) CILLANIC ACID was filtered and theisolated wet solid was stirred for 8 To an enzyme Solution 8-: activity8-) m at H 11,5421 i i ld aqueous cyanogen b obtained as in Example 2cwas added benzylpenicillin as mide (5%, 2 ml.). The solid gel wasrecovered by filtra- Potassium Salt dissolved in Water tion and washedwell on the filter with ice-water and ice- The Solution Was incubated at37 C. and the pH kept 1d 1 M borax i was h Suspended i 1 M borax at 7.8by the periodic addition of 2.5 N sodium hydroxide. (4 L) d i d i h E liacylase ti it 159,000 40 After 6 hours the solution was cooled to 10 C.and exu./g.; 0.106 g.), obtained according to Example 7d, for 24 tradedat P 2 With melhylisobutylketofle hrs. at +4 C. The mixture was filteredand the recovered remove the residual benzylpenicillin and thephenylacetic Solid material was Washed thoroughly on h filt i h acidside chain, which is split off during the formation of water to give 1.2g. of wet polymer with an specific activity the 6-aminopenicillanicacid. After the extraction the of jg. water solution was separated fromthe methylisobutyl (b) Sephadex G 25 (0.100 g.) was stirred for 8 min.keioneat pH 11.5-12 with ice-cold aqueous cyanogen bromide The pH of thewater solution was adjusted to 7.5 and 5 2 1 d filt the volume reducedto 110 ml. in vacuo. The concentrated The recovered gel was washed wellwith ice-water and solution was cooled to 5 C. and filtered. When thefiltered ice-cold 0.1 M borax (4 ml.). E. coli acylase (activitysolution was acidified to pH 4.3 vn'th 5 N hydrochloric 159,000 u./g.;0.106 g.) obtained according to Example acid the crystalline6-aminopenicillanic acid precipitated. 7d, was added and the mixture wasstirred for 24 hrs. at After filtration the crystals were washed twicewith cold +4 C. Then the mixture was adjusted to pH 7.5 and fil- Water(5 ml.) followed by dry acetone (twice with 5 ml.) tered. The solidmaterial was washed well on the filter and dried in vacuo to give ayield of 8.51 g. of 6-aminowith water to give 0.3 g. of polymer with anspecific acpencillanic acid. The purity of the product was 99% andtivity of 212 u./ g. the total yield calculated on the benzylpenicillinwas 69%.

-(c) Experiment b was repeated with DEAE-Sepha- In analogous way6-aminopenicillanic acid was predex A 50 (0.1 g.) instead of Sephadex G25. The yield pared using enzyme preparations prepared according to was1.25 g. of wet polymer with a specific activity of the precedingexamples. Yield and purity of the acid ob- 435 tained is given in thefollowing Table I.

gTABLE I Enzyme Benzyl- Geminopenicillanic acid penicillin Preparedpotassium asin Amount, Activity, Yield, Purity, Yield, salt, g.Exampleg. u./g. g. percent percent 21 a e. 3 1,600 10. 1 9s. 2 s2 21 4a.4. 2 2, 920 9. s 96. 0 78 500 4b 450 800 261.3 93.5 as 21 s 5. 4. 2, 3407. 5s 97. 0 21 7a 145 87 9.9 100 81 2,100 7b 780 1,610 1, 064 100 88 217 0.1 123, 000 9.3 98.0 21 7d 0.11 116,500 10.35 as 21 9b 5. 4 2, 400 9.1 100 75 21 10 2. 4 6, 000 8. 82 100 72 21 9a 0. 9 14, 000 9. 2a 100 76Corrected for purity.

EXAMPLE 15PREPARATION OF p-NITROBEN- ZYLESTER OF 6-AMINOPENICILLANICACID An enzyme solution (18.6 g., activity 1610 u./g.) obtained as inExample 7b was diluted with water (700 ml.)

14 The sodium salt of 6-(D-a-azido-fenylacetamido)- penicillanic acidwas filtered. The crystals were washed with 50 ml. methylisobutyl ketone(50 ml.) and dried in vacuo at 50 C. to give a yield of 16.5 g. Thepurity of the product was 96% and the total yield calculated on the andmethanol (120 ml.) and adjusted to pH 7.8 by the 5 benzylpenicillin was75%. addition of 0.1 molar sodium hydroxide. A solution of p- Inanalogous way 6-(D-a-azido-fenylacetamido)-penI- nitrobenzylbenzylpenicillinate (3.1 g., 6.6 mol) in methcillanic acid sodium saltwas prepared using other enzyme anol (60 ml.) was added. The mixture wasstirred at 37 preparations prepared according to the preceding exam- C.and kept at pH 7.8 by the addition of 0.1 molar sodium ples. Yield andpurity of the penicillin is given in the folhydroxide. After 2.5 hoursthe mixture was cooled and exlowing Table II.

TABLE II G-(D-a-azido-feny1acetamido)- Benzyl- Enzyme penicillanie acidpenicillin potassium Preparedas Amount, Activity, Yield, Purity, Yield,salt, g. in Exampleg. u.g. g. percent percent 21 3 6.3 1, 600 18. 9 95.8 s2 21 4a 4. 2 2, 920 18. 94. 0 71 21 5 5. 4 2, 340 16. 2 s9. 7 00 217a 145 87 20. 4 95. s s5 21 7b 0 1, s90 19. 7 9e. 0 s7 21 70 0.1 128,00018.5 90.0 76 21 9b 5. 4 2, 400 20. 5 92. 7 s9 21 10 2. 4 0, 000 18.5 95.5 75 21 9a 0. 9 14, 000 20. 0 91. 4 89 *Oorreetedforpurity.

tracted with ethyl acetate (500 ml.). The aqueous layer EXAMPLE17ENZYMATIC SYNTHESIS OF was separated and extracted with an additionalportion BENZYLPENICILLIN of ethyl acetate (250 ml.). The organic layerswere com- AP A (175 mg and h 1 p eny acetic acid (111 m bmed and Wlthanhydvus sofhum gl eg q was dissolved in potassium phosphate buffer (10m l.) hr. After filtration the orgamc solution was ivi e in 0 at pH 70.Acylase prepared as in Example 7c (35 mg two equal parts'.one.wasconc?ntrated m vacuo at activity 128,000 u./g.) was dissolved in Water(5 ml.) temperature to give the free p-nitrobenzylester of "6-arn1noandadded to the first prepared Solution pencillanic acid as an oily residue(1.1 g.). The product Thc pH was adjusted to 50 with M H31304 and thewas found to Contam Its IR'SPectmm bands at 3250 40 solution wasincubated at 37 C. The pH was kept at 5.0 51252 3 2 23 iig gggg gggggzzg NHz'group and after 4 hours the solution was cooled and analyzedBenzenesulphomc acid (0.58 g.) dissolved in acetone g zg ig ggg i i sfig 30% of the 6 APA was trans (20 ml.) was added to the other part ofethyl acetate solution which then was concentrated in vacuo to a volumeEXAMPLE 18-FIXING OF PENICILLIN ACYLASE of about 30 ml. On addition ofether (10 ml.) and stand- TO A POLYMER CARRIER ing over night in the icebox the benzenesulphonic acid salt of p-nitrobenzyl 6-aminopenicillinate(0.8 g.) def t i G (0.5 gt) Wafs aditiled to water (20 ml.) posited aswhite crystals, M.P. 148-151" C., identical with the room g 3 fee aThereafter the product described in British patent application No. i um9 e an a co d Solutlon cyailogen 33 734/67 Example 4. bromide (1 g.) inether (15 ml.) was added with stirring.

The pH was kept at 11.512 by addition of sodium hy- EXAMPLE 16PREPARATION OF droxide. After the reaction had eeased the mixture wasFENYLACETAMIDO) PENICILLANIC ACID filtered and the wet polymer washedwith ice-water and finally with ice-cold 0.1 M borax solution.

T an enzyme l i (233 g, activity 42 was The wet polymer was added to asolution of penicillin dd d a l i of b l i u 20 g.) as Potas acrylase(10 m1., activity 400 u./ml.) followed by solid sium alt i at (317 borax(380 mg.) and the mixture was stirred at +4-5 C.

The mixture was incubated at 3 C. and the PH kept for 24 hours.Thereafter the sephadeX-acylase compound at 7.8 by periodic addition of2.5 N sodium hydroxide. was filtered and Washed With Water giving 8- ofWell After 6 hours the reaction mixture contained 10.7 g. of substancewith an activity of 37501118- 31322323522 :22. .0 .H 0F ACYLASE 3.0 withlQ N sulphuric acid. Methylisobutylketone (300 To A POLYMER CARRIER ml.)containing D-a-azidophenylacetyl chlorine (62 mm Sephadex G 200 250 w ddd was added during stirring. The stirring was continued 30 65 liters)and left at room t emperature f0: flir minutes and pH was kept at 3.0 byperiodic addition of 2.5 Thereafter the mixture was cooled and 5 Nsodium by N sodium hydroxide. The reaction mixture was filtered droxide(6 liters) was added. With vigorous stirring through Cehte 505 (10 g.)and the methylisobutyl ketone cyanogen chloride (320 g.) was conveyedinto the mixwas separated from the water phase. h ture at 0-3 C. duringa period of 1.5 hours. Hyflo Super The methylisobutyl ketone solutionwas dried with Gel (700 g.) was added and the mixture filtered andwater-free sod um sulphate (25g washed with cold water (40 liters)followed by cold 0 1 liafter filtation the6-(D-ot-3ZldO-f8l1YlflCCt3Il1id0) -peni- M borax (20 liters). I ci anicaci was precipitated as sodium salt by addition The wet substance wasadded to a solution of peniof 41 ml. of a 2 N solution of the sodiumsalt of 2-ethyl- 0111111 acylase (3,000 m1. contain 6550 u./ml.)followed caproic acid in methylisobutyl ketone.

by borax 9.). The mixture was stirred for 24 hours at +45 C. whereuponthe polymer was filtered and washed with water giving 5072 of wetsephadex-acylase with an activity of 1245 u./g. Penicillin acylase notcoupled in the reaction could be almost quantitatively recovered fromthe mother liquor by desalting on Sephadex G 25 and concentrating of thesolution in vacuo. This solution could be used directly in a newcoupling reaction.

EXAMPLE -PREPARATION OF G-AMINO- PENICILLANIC ACID Sephadex-acylase,prepared according to Example 19, (17 g., activity 1245 u./g.), wassuspended in water (600 ml.), boric acid (2.2 g.) added followed bybenzylpenicillin potassium salt (21 g.) in water (100 ml.). The

mixture was stirredat 35 C. and the pH'kept constant at 7.0 by additionof 0.5" N sodium hydroxide. After 3 hours the reaction mixture,containing 11.7 g. of 6- aminopenicillanic acid ('6APA) (96% wasfiltered and the solution extracted with a half volume ofmethyl'isobutyl ketone at pH 3 by addition of 5 N hydrochloric acid inorder to remove unreacted penicillin and phenylacetic acid split ofiduring the reaction. The water solution was separated, adjusted to pH7.5 by addition of sodium hydroxide and concentrated in vacuo to avolume of about 120 ml.

The solution was cooled to 5 C. and acidified with stirring to pH 4.3 byaddition of 5 N hydrochloric acid in order to precipitate crystalline6-APA. After 1 hour the crystals were filtered, washed with cold waterfollowed by dry acetone and dried in vacuo to give 6-aminopenicillanicacid (10.6 g., purity 99%, total yield 86%).

EXAMPLE 2l-PREPARATION OF 6-(D-u-AMINO- PHENYLACETAMIDO) PENICILLANICACID Sephadex-acylase (260 g., activity 1500 u./g.) was suspended inwater (9 liters), NaI-I PO 2 aq. (91 g.) in water (1 liter) adjusted topH 7.0 with sodium hydroxide, added, followed by benzyl penicillinpotassium salt (350 g.). The mixture was incubated at 35 C. withstirring and the pH kept constant at 7.0 by addition of 0.5 N sodiumhydroxide. After 165 minutes the mixture was filtered and the solutioncontaining 195 g. of 6- arninopenicillanic acid was cooled to 9 C. andacidified to pH 3.0' with 5 M sulphuric acid. Methyl isobutyl ketone(5.3 liters) was added followed by a solution of D-a-azido-phenylacetylchloride (0.96 mol) in methyl isobutyl ketone (400 ml.) during a periodof minutes. The pH was kept at 3.0 by periodic arrition of 2.5 N sodiumhydroxide (1100 ml.) and the stirring continued for an additional 20min. period. The organic layer was separated, filtered with Celite 505(100 g.), cooled and extracted at pH 7.0 by addition of 15% potassiumhydroxide (225 ml.). The water phase was separated and the organic layerextracted with an additional amount of 15 KOH (15 ml.). The Watersolutions of 6-(D-0cazido-phenylacetamido)penicillanic acid were pooledand adjusted to 1.35 liters by addition of water.

Raney nickel g.) was suspended in water and hydrated at 60 p.s.i. and 20C. for 30 minutes. Thereafter the penicillin solution was added duringcooling in icewater and hydrated at 60 p.s.i. and 20 C. for 30 minutes.The catalyst was filtered and the solution rapidly cooled and adjustedfrom pH 9.8 to 2.0 by addition of 5 M sulphuric acid. The solution wasextracted with methyl isobutyl ketone (1650 ml.) in the cold, the waterphase separated and adjusted to pH 4.5 with 15% KOH (280 ml.). Afterstirring in ice-water bath for 2 hours the crystalline substance wasfiltered, washed with cold water and dried in vacuo overnight to give201 g. of 6-(D-a-aminophenylacetamido)penicillanic acid. Purity 98.2%(as trihydrate), yield 52%.

EXAMPLE 22--PREPARATION OF SODIUM 3-0- CHLOROPHENYL 5METHYL-4-ISOXAZOLYL PENICILLIN 6-aminopem'cillanic acid (84 g.) preparedas in Exam- 16 ple 3 was suspended in water (1200 ml.), the mixturecooled to 5 C; and pH adjusted t07.5 by addition of 10% sodium hydroxide(123 ml.) with stirring. Methyl isobutyl ketone (1200 ml.) was addedfollowed by 3-o-chlorophenyl-S-methylisoxazole-4-carbonyl chloride (120g.) in methyl isobutyl ketone ml.). The stirring was continued for 2hours at 22-27 C. The organic layer was separated and dried with 60 g.ofanhydrous sodium sulphate. Anhydrous sodium acetate (37.2 g.) wasadded to the clear organic solution and the mixture stirred for twohours at room temperature. The crude penicillin salt was filtered,washed with methyl isobutyl ketone and dried in vacuo overnight. Theyield was 171.6 g. The crude sodium salt was recrystalized from water(170 ml.) and isopropyl alcohol (170 ml.) by addition of anhydrousisopropyl alcohol (2170 ml.). The mixture was stirred for 4 hours, thecrystalline substance filtered and washed with isopropyl alcohol anddried in vacuo overnight, to give 145.1 g. of sodium3-o-chlorophenyl-5-methyl-4-isoxazolyl penicillin, purity 97.9%, totalyield 76.5%.

EXAMPLE 23ANTIGENICITY TESTS Method: Passive cutaneous anaphylaxis inguinea pigs essentially according to de Weck et a1. Int. Arch. Allergy33 (1968) 535-567.

Rabbit 6-APA antiserum was produced by repeated subcutaneous injectionsof crude 6-APA plus two injections by the same route of a retentate froma dialyzed ,6- APA batch. In passive hemagglutination tests thisantiserum showed a titre of 1/4096. One ml. of this 6-APA antiserum wasinjected intravenously ina white guinea pig. Twenty-four hours later 0.1ml. of a 5% solution in saline of Evans blue was administeredintravenously followed 1 hour later by intradermal administration of 0.1ml. containing 4 mg. of 6-APA prepared according to Examples 14d, 14g,and 14i, respectively. On a fourth site on the back of the animal asample of 6-APA prepared in a conventional Way using whole cells of E.col'i was likewise given. The 6-APA samples were injected immediatelyupon their dissolution. As control 0.1 ml. of normal saline was injectedintradermally. In a second guinea pig the same procedure was repeatedexcept that the 6-APA antiserum was replaced by 1 ml. of normal salinegiven intravenously.

Two hours after the intradermal injections the extent of blueing at theinjection sites was determained and expressed as the product of twoperpendicular diameters.

Results TABLE III The product of two perpendicular diameters (mm?) ofblue spots caused by injection of 4 mg. intradermally of compoundsindicated 7 Non- I sensitized sensitized Difanim animal ference Preparedby use of E. coli cells 256 146 6-APA according to Example N o Theresults show that a sample of 6-APA prepared by conventional methodsdiffers from the other sample of 6-APA with regard to ability to elicitthe passive cutaneous anaphylactic reaction. This is an expression ofreduced antigenicity in 6-APA according to the present invention ascompared to 6-APA prepared by conventional procedures, using whole cellsfrom E. coli.

EXAMPLE 24-DETERMINATION OF PROPERTIES WITH 6AMINOPENICILLANIC ACID ANDPENI- CILLINS PREPARED USING POLYMER-FIXED PENICILLIN ACYLASE (A)Penicillin immunogenicity (antibody value) (B) Contents ofhigh-molecular impurities (C) Contents of amino acids (proteins).

The following methods were used.

(A) Determination of penicillin immunogenicity (antibody value)Principle.Dialyzis retentate is given to three albino rabbits as threeweekly injections by the subcutaneous route. Blood samples are collectedone week after each injection and two weeks after the last one. Theamount of antibodies in the blood samples is determined, and isexpressed as antibody number which is the sum of all the individualtitre values. The higher the antibody number the more immunogenic is thepenicillin preparation.

Preparation of dialyzis retentate of the tested penicillins and6-aminopenicillanic acid is carried out as follows:

A solution of sodium 3 o-chlorophenyl-S-methyl-4- isoxazolyl penicillin(10 g.) in deionized water (200 ml.) is transferred to a semipermeabletube and dialyzed against flowing water during five days. Thereafter,the retentate is concentrated in vacuo to a volume of 10 ml. Solutionsof 6-aminopenicillanic and 6-(D-a-aminophenylacetarnido) penicillanicacid are dialyzed and concentrated in the same manner. For dissolvingthese substances, however, sodium hydroxide must be added.

Immunization.-Dialyzis retentate of the penicillin sample is emulsifiedwith Freunds complete adjuvant. The mixture is injected subcutaneouslyin the rabbit in an amount corresponding to 250 mg. of penicillin.Injections are made at day 0.8 and 15. Three rabbits are treatedidentically.

Blood is collected at day 8, 15, 22 and 29. Serum is prepared and testedfor the presence of antibodies.

Antibody determination-The amount of anti-penicillin antibodies presentin the serum sample is determined by a passive hemagglutinationtechnique described by Thiel et al., J. Allergy 35 399 (1964). Thehighest serum dilution giving agglutination of penicilloylated red bloodcells is determined and is taken as the antibody titre of thatparticular serum sample. The titre values of twelve such serumsamples-obtained from the three rabbits participating in the test-areadded to make up the antibody value. The antibody value is thus anexpression of the potency of the penicillin preparation as an immunogen.

(B) Separation and determination of high molecular impurities inpenicillins by gel permeation chromatography (GPC) An aqueous solutionof the penicillin is run on a GPC- column (Sephadex G 25). Proteins andother high molecular impurities are eluted with the void volume whilethe penicillin is retarded. The UV-absorption is continuouslyregistered.

Apparatus-Chromatography column x 100 cm.; Uvicord I with recorder, LKBProducts; peristaltic pump; pH-meter. I

Reagents-Sephadex G 25 Fine, Pharmacia Fine Chemicals; 5 N sodiumhydroxide.

Column.--330 g. Sephadex G 25 Fine is allowed to swell in excess ofpurified Water for 24 hours. The column is packed according to themanufacturers instruction. 1 Procedure-10 g. of penicillin is dissolvedor suspended in 35 ml. of purified water. In case of not soluble in thisvolume 5 N sodium hydroxide is added drop-wise under pH control andvigorous stirring until the-penicillin is dissolved. pH is kept below9.0 under this procedure.

The penicillin solution is transferred to the column and chromatographedwith water as eluant at a flow rate of l-2 ml. per minute. The columnshall be run with ascendfi The UV-absorption of the eluate at 254 nm. isrecorded (Uvicord) The absorbance of the high molecular fraction in thevoid volume is measured.

Determination of contents of amino acids in penicillin preparationsDialyzis retentate of 10 g. penicillin'is evaporated-to dryness at atemperature of not more than 40 C. The retentate is transferred to atest'tube containing10 ml. 6 N HCl. The sample solution obtainedis'freezed and the test tube thereafter evacuated and sealed by heating.The sample is hydrolyzed at C. for 22 hours whereafter it is evaporatedto dryness and thereafter redissolved in 0.2 N sodium-citrate buffer ofpH 2.2. The content of amino acids in the sample is thereafter analyzedby Stein-Moore technique using a Beckman type 1200 amino acid analyzer.For analysis of basic amino acids resin Aminex A-5 and 0.35 Nsodium-citrate buffer, pH 5.25, is used. For analysis of acidic andneutral amino acids resin Aminex A-4 and 0.2 N sodium-citrate buffer, pH3.25 respectively 0.2 N sodium-citrate buffer, pH 4.25, is used. Theanalysis is carried out at a temperature of 50 C. The content of proteinin the tested penicillin preparation is calculated by adding the amountsof amino acids determined and is given in terms of weight, parts permillion, ppm.

The results of the tests described under A, B, and C above on6-aminopenicillanic acid, 6-(D-a-aminophenylacetamido)penicillanic acid,and 3-(o-chlorophenyl)-5- methyl-4-isoxazolylpenicillin are collected inTable IV below. Test results on 6-arninopenicillanic acid prepared frompenicillin G using polymer-fixed penicillin acylase from E. coliaccording to the invention are compared with test results on6-aminopenicillanic acid prepared from penicillin G using cellsuspensions of E. coli cells according to previous technique. In thesame way, test results on 6-(D-waminophenylacetamido)penicillanic acidand 3 (o-chlorophenyl)-S-methyl-4-isoxazolylpenicillin, prepared using6-APA obtained according to the present invention, and using 6-APAobtained with E co l! cell suspensions, are compared.

TABLE IV Immunogenicity (antibody value) contents of highmolecularweight impurities, and contents of amino acids (proteins), inpreparations of 6-APA, 6-(D-a-aminophenylacetamido)penicillanic acid and3-(o-chlorophenyl) -5-methyl-4-isoxazolylpenicillin a High Contentsmolecular of amino impurities I acids (absorb-ance antibody Substance(p.p.m.) at'254 mm.) value fi-(Dflaminophenylacetamido) 54 0. 030 l7penleillanic acid prepared using 178 0.025 26 unisolated 6 APA preparedvia 178 0. 025 25 E. coli cell suspensions. 148 O. 030 31 70 0.035 26tl-(D-a-aminophenylacetamido) 17 0.015

' pencillanic acid from unisolated ti-APA prepared using poly mer-fixedacylase.

3(o-ehlorophenyl)-5-methyl-4 11 0. 155 4 isoxazolylpenicillin irom iso-11 0.120 8 lated 6-APA prepared via E. coli cell suspensions. 3-(o-chlorophenyl) -5-methyl-4 2 0. 035 2 isorazolylpenicillin trom 6-APAprepared using polymer-fixed acylase.

One reason for the occurrence of such allergic reactions which may occurat therapeutical use of penicillins is the presence of proteinaceousimpurities in the preparations administered to the patients. As isevident from the test results reported in Table IV, the amount ofproteins and other high molecular weight impurities in penicillinando-APA-preparations is significantly reduced when polymer-fixedpenicillin acylase according to the present invention is used. It isalso evident from Table IV that the antibody value of the testedpreparations is sharply reduced in the preparations prepared usingpolymer-fixed penicillin acylase in comparison with the preparationsprepared using previously known preparations of penicillin acylase,which means that use of the polymer-fixed penicillin acylase results inpreparations of penicillins and 6-APA with less tendency to produceallergic reactions atadministration to patients. Thus it is establishedby three independent methods that use of polymer-fixed penicillinacylase in the preparation of 6-aminopenicillanic acid results inmarkedly improved properties with regard to hypoallergenicity in the6-aminopem'cillanic acid obtained and in semisynthetic penicillinsprepared from such 6-aminopenicillanic acid.

We claim:

1. A continuous process for the production of hypoallergenic6-aminopenicillanic acid by enzymatic degradation of an enzymicallydeacylablc natural penicillin with an Escherichia coli penicillinacylase, which process comprises reacting the penicillinwith apenicillin acylase preparation consisting of cell-free Escherichia colipenicillin acylase chemically fixed to a polymeric support, saidpenicillin acylase preparation being obtained by v (a) fermenting anEscherichia coli strain producing intracellular penicillin acylase;

(b) simultaneously separating the cell material from the culture brothof the microorganism and releasing the intracellular penicillin acylaseform the cell material in a self-cleaning centrifugal separator byapplying to said cell material a centrifugal force corresponding to apressure of about 500 p.s.i. to 2,000 psi. at a temperature ranging fromabout 0 C. to 50 C. and intermittently expelling the cell materialthrough a peripheral slit;

(c) stirring the expelled cell material in aqueous solutionat atemperature ranging'from' about 10 C. to

50 C. for about 10 to 5.0 hours to dissolve the acylase relased in step(b);

(d) subsequently extracting the acylase from the in- I active cellmaterial of the aqueous solution; and

p (e) coupling chemically of the extracted acylase to an activatedpolymeric material.

2. A process according to claim 1, wherein the penicillin is benzylpenicillin,

3. A process according to claim 1', wherein the activated polymericmaterial used in step (e) is a polysaccharide treated with a cyanogenhalide.

4. A processaccording to claim 3, wherein the activatedpolymericmaterial used in step (e) is a polysaccharide treated withcyanogen bromide.

5. A process according to claim 3, wherein the activated polymericmaterial used in step (e) is a polysaccharide treated with cyanogenchloride.

6. A process according to claim 3, inclusive wherein the polysaccharidevi's equilibrated with water and thereafter reacted in an aqueous mediumwith the cyanogen halide at'a temperature of from about 0 C. to about 25C. during a time period from about 2 to about 30 t 5 hours at a pH of8.5-9.5.

7. A process according to claim 1 wherein an aqueous suspension of theactivated polymeric material and the natural penicillin is incubated-ata temperature of from about 30 C. to about 45 C. at a pH of 6.5-8.2until the enzymatic reaction has ceased.

8. A process according to claim 1, wherein in step (b) a centrifugalpressure of 9004100 p.s.i. is applied to the cell material atatemperature ranging from 15 C. to 40 C., and wherein the cell materialis intermittently expelled within 0.05 to 1.0 sec. through a peripheralslit having a width of 0.1 to 1.5 mm. 9. A process according to claim 1wherein the extracted enzyme is precipitated from the cell-free aqueoussolution at pH 4 to 6 with tannin in the presence of chelating agentsforming complexes with iron ions.

10. A process according to claim 1 wherein the culture broth issaturated with an organic solvent having low solubility in waterselected from the group consisting of butylacetate, isobutylacetate, andamylacetate.

11. A process according to claim 1 wherein the expelled cell material isstirred in an aqueous solution containing 1.05.0% water immiscibleorganic solvent selected from the group consisting of methylisobutylketone, butylacetate, isobutylacetate, amylacetate, benzene, toluene andchloroform.

12. A process according to claim 11 wherein the pH of the aqueoussolution is maintained between 6.5 and 9.0 by adding a base selectedfrom the group consisting of sodium hydroxide, potassium hydroxide,ammonia, triethylamine and N-ethyl-piperidine. I

13. A process according to claim 1 wherein any remaining inactive cellmaterial is precipitated from the aqueous s7o(l]ut8io5n to pH 3.0-6.0and thereafter adjusting the pH to 14. A process according to claim 1wherein the extracted acylase is concentrated and isolated by passingthe aqueous acylase solution at pH 3.5-6.0 through a column of acationic ion-exchanger and releasing the acylase' by elution at pH6.0-8.0 with a weak buffer solution containing an organic salt selectedfrom the group consisting of ammonium acetate andtrishydroxymethylammomum acetate.

15. A process according to claim 1 wherein the extracted acylase isisolated from said aqueous solution in the solid state by known dryingtechniques.

References Cited UNITED STATES PATENTS 3,297,54 1/1967 Johnson et a1.-36P 3,666,627 5/1972 Messing 195Dig. 11 ALVIN. E. TANENHOLTZ, PrimaryExaminer US. or. X.R.

195-66, Dig. 11; 424 271

